Isopropanol, as an industrial solvent for paints, inks, or the like, or as an industrial raw material for various uses, is currently produced in an amount of about 1,800,000 tons per year worldwide and about 180,000 tons per year in Japan. Also, isopropanol can be converted into propylene by a simple dehydration reaction, and therefore can be used as a source of polypropylene, which is currently produced in an amount of about 3,100,000 tons per year in Japan.
However, these products are all derived from fossil crude resources.
Development of new methods for producing energy or chemical products not from fossil resources, almost all of which are imported, but from renewable resources is strongly desired in order to solve global environmental issues such as global warming, exhaustion of fossil resources, and soaring oil prices, and to reduce the dependence on foreign countries for important raw material resources of chemical products. Efficient technology for producing isopropanol from renewable resources, such as biomass, would be one of the measures to solve these problems.
As an example of microbial production of isopropanol from biomass resources, it is reported that a kind of Clostridium that performs acetone-butanol fermentation produces isopropanol in addition to butanol (isopropanol-butanol fermentation). This is because the Clostridium exhibiting such a fermentation pattern has isopropanol dehydrogenase, which reduces acetone to isopropanol as a catalyst.
In recent years, the production and use of biofuel is increasing around the world, and from the standpoint of biofuel production, researches on butanol production based on acetone-butanol fermentation are attracting attention again. However, these researches are primarily intended for butanol production, and few of them are intended for isopropanol production.
As bacteria which produce isopropanol, Clostridium bacteria known as isopropanol-butanol fermenting bacteria, such as Clostridium beijerinckii, Clostridium aurantibutyricum, etc. have been reported so far (Applied and Environmental Microbiology, Vol. 45, 1983, 1160-1163).
However, isopropanol production using Clostridium bacteria has the following problems.
(1) In isopropanol-butanol fermentation by Clostridium bacteria, butanol is the main fermentation metabolite, and isopropanol is produced in low efficiency. (The ratio of isopropanol/butanol to be produced is about ⅕ to 1/10.)
(2) Clostridium bacteria require strictly anaerobic conditions in proliferation and in production of isopropanol. Therefore, for such strictly anaerobic conditions, complicated culture procedure involving, for example, replacement of the air in the culture apparatus with an inert gas such as nitrogen gas, is required. In addition, the proliferation rate is extremely low, and as a result, the isopropanol production rate is low. To solve these problems, use of aerobic bacteria with a high proliferation rate may be considered, but no microorganism (an aerobic bacterium or a facultative anaerobic bacterium) capable of producing isopropanol with high efficiency and proliferating under aerobic conditions has yet been known.
(3) In isopropanol-butanol fermentation, acetic acid and butyric acid are generated during cell-growth phase, and during stationary phase, in which cell growth stops, acidification to lower pH in fermentation culture triggers transition to solvent (isopropanol and butanol)-production phase, resulting in a drastic change in metabolic system (catabolic shift) and production of isopropanol and butanol. Thus, fermentation process must be strictly controlled, and substantial time is required from the start of fermentation to production of isopropanol and butanol. Also, these Clostridium bacteria have problems including that transition to sporulation phase stops the production of isopropanol and butanol, that is, the production of isopropanol does not last long.
To solve these problems, inventions of a novel isopropanol-producing microorganism and a novel isopropanol-producing process have been desired.
For producing isopropanol with the use of Clostridium bacteria, the following techniques have been disclosed.
Applied and Environmental Microbiology, Vol. 45, 1983, 1160-1163 discloses that Clostridium beijerinckii produces isopropanol in addition to butanol and that Clostridium aurantibutyricum produces isopropanol in addition to butanol and acetone.
Also, Enzyme and Microbial Technology, Vol. 5, 1983, 46-54 and Biotechnology and Bioengineering, Vol. 39, 1992, 148-156 disclose a continuous isopropanol-producing technique using immobilized Clostridium bacteria; Applied Microbiology and Biotechnology, Vol. 32, 1989, 22-26 discloses an isopropanol-producing technique using agglutinating Clostridium bacteria; Applied Microbiology and Biotechnology, Vol. 25, 1986, 29-31 discloses a technique of catabolite repression in isopropanol-butanol fermentation mediated by Clostridium bacteria, by adding a polymer resin to adsorb catabolites, which are isopropanol and butanol. However, the focus of these techniques is producing butanol, and they are all isopropanol-producing techniques using Clostridium bacteria under anaerobic conditions. Therefore, they are not fundamental solutions to the problems pointed out in the above (1), (2), (3), etc.
Meanwhile, although not for isopropanol production, the following acetone-butanol producing techniques using Clostridium bacteria have been disclosed so far.
WO 2006/007530 discloses a technique of controlling a gene responsible for sporulation to delay sporulation phase for increasing butanol production; US 2005/089979 A1 and Bioprocess and Biosystems Engineering, Vol. 27, 2005, 207-214 disclose a technique of continuous extraction of butanol by the gas-stripping method in continuous fermentation; Pakistan Journal of Biological Sciences, Vol. 9, 2006, 1923-1928 and Applied Biochemistry and Biotechnology, Vol. 113-116, 2004, 887-898 disclose a butanol-producing technique by immobilizing Clostridium bacteria; Journal of Biotechnology, Vol. 120, p 197-206 discloses a technique of recycling bacteria cells in continuous fermentation by using high-density Clostridium bacteria. Although these techniques are considered to be applicable to isopropanol-butanol fermentation as well using Clostridium bacteria, they are nothing but production techniques using Clostridium bacteria under anaerobic conditions, and therefore not fundamental solutions to the above-mentioned problems.
Examples of techniques for producing isopropanol with the use of bacteria other than Clostridium bacteria include the following.
The inventors have already proposed a technique for producing isopropanol with the use of Escherichia coli as a host (JP 2007-222633 A and Applied Microbiology and Biotechnology, Vol. 77, 2008, 1219-1224). Applied and Environmental Microbiology, Vol. 73, 2007, 7814-7818 discloses a technique for producing isopropanol by expressing genes which encode acetyl-CoA acetyltransferase, acetoacetyl-CoA:acetate-CoA transferase, acetoacetate decarboxylase, and isopropanol dehydrogenase derived from a microorganism selected from Clostridium acetobutylicum, Escherichia coli, Clostridium beijerinckii, and Thermoanaerobacter brockii, in Escherichia coli as a host.
However, the above-mentioned techniques have room for improvement to produce isopropanol with the use of a microorganism more efficiently.